Many trees and woody horticultural species, such as loblolly pine, Radiata pine, and eucalyptus, have lengthy breeding cycles. For this reason, using traditional breeding programs to incorporate new and commercially desirable traits into those trees and woody species is time-consuming and cumbersome. It takes too long, or it is not feasible, for instance, to back-cross trees in order to introduce a new genetic trait into a desired line. Furthermore, it is often difficult to ramp up production of those trees and species to levels that are suitable for large-scale propagation, by simply employing conventional vegetative or clonal propagation methods, including those frequently used for somatic embryogenesis.
This is largely because conventional vegetative and clonal propagation strategies, particularly somatic embryogenesis strategies, require numerous manual handling steps that involve physical transfer of embryos from one gelled surface to another. The starting explants, e.g., seeds, are typically placed onto a Petri dish with medium that contains plant growth regulators and nutrients for 6-8 weeks, until embryogenic tissue forms. The tissue is then bulked up on either a gelled or liquid maintenance medium to obtain sufficient mass for subsequent use. The resultant embryogenic tissue is then transferred to another Petri dish that contains a maturation medium to promote formation of somatic embryos. Embryos are then conditioned until they are ready for germination. When the germinated embryos have grown large enough, they are then transferred to a greenhouse and eventually planted in the field.
This conventional gel-based Petri dish methodology is cumbersome and time-consuming and does not lend itself to routine automation or commercial scale-up. Hence, under the traditional system, large-scale production typically relies on manual labor, which can prove to be expensive. Approximately sixty-percent of costs involved in large-scale propagation, for instance, are attributable to man-power involved in “rooting” efforts. See Mass propagation of conifer trees in liquid cultures—progress towards commercialization. In: Hvoslef-Eide A. K. and W. Preil (eds.) Liquid culture systems for in vitro plant propagation. Springer. Netherlands, pages 389-402, 2005.
Gel-based culturing systems are not readily amenable to automation since they require manual intervention. By contrast, liquid culturing systems for somatic embryogenesis can be automated, which makes it easier and more efficient to handle and change liquid media. Indeed, tissue and cell transfer, sub-culturing, and harvesting can all be accomplished efficiently in a liquid culture systems. See Hvoslef-Eide A. K. and W. Preil (eds.) Liquid culture systems for in vitro plant propagation. Springer. Dordrecht, The Netherlands, 2005.
Large-scale “bioreactor” vessels, which propagate cell and tissue cultures in large volumes of liquid, therefore, are useful for maintaining and bulking-up embryogenic cells and tissues (Hvoslef-Edie and Preil 2005, supra). The problem is that these large-scale bioreactors and non-gel culturing systems have not proven adaptable for producing conifer somatic embryos.
Other systems have therefore evolved that employ an intermediate step of culturing conifer tissues on membrane rafts that are floated on liquid medium. Such systems, often referred to as “temporary immersion” systems, however, prove costly and complex when they are adapted to perform at a large scale levels for conifer somatic embryogenesis. See Vagner et al., in Hvoslef-Eide A. K. and W. Preil (supra) at pages 295-302.
Another issue concerning large-scale production of conifer somatic embryos is the use, conventionally, of polyethylene glycol in development media. It is well accepted that a relatively high concentration of polyethylene glycol in gel-based development medium is a routine and standard method for stimulating embryo development and increasing embryogenic cultures in conifers. For instance, Gupta used highly concentrated amounts of polyethylene glycol (PEG), such as 10% to 18%, in both gelled, i.e., “solid,” and liquid embryo development media for development of Douglas fir somatic embryos. See U.S. Patent Application Nos. 2005/0003415, 2005/0026281, 2005/0188436, and 2005/0198713, and U.S. Pat. No. 5,036,007. In those systems, absorbent pads were soaked with liquid development medium and embryogcnic tissue placed on the pads.
Similarly, Attree et al., Ann. Bot. 68:519-525. 1991, observed a 3-fold increase in the maturation frequency of white spruce somatic embryos on gel-based development media containing an optimum of 5% to 7.5% polyethylene glycol. Attree found that embryo development and production was not as effective when lower concentrations of polyethylene glycol were used in the gel media.
Attree later reported on a technique for transferring embryogenic suspension cultures of Picea glauca onto an absorbent pad, which was in contact with liquid embryo development medium, but with the surface of the pad above the liquid medium surface. See Attree et al., Plant Cell Reports. 13:601-606. 1994. Hence, the embryos were exposed to the atmosphere inside the chamber, rather than directly in the liquid. Under Attree's system, 6,314 cotyledon stage somatic embryos were harvested from one chamber using 3 liters of medium in 7 weeks (from 10 grams of embryogenic starting tissue grown from a liquid culture). This equates to just about 2 somatic embryos per ml of liquid embryo development medium used in the chamber.
In U.S. Pat. No. 6,340,594, Attree developed a continuous-flow solid-support bioreactor. In this system, the embryogenic tissue and resulting embryos were not submerged in the liquid medium. Hence, embryo development did not occur completely in liquid medium.
Likewise, Paques also reported that only those conifer Picea abies embryos that were directly in contact with the atmosphere, as opposed to those submerged in liquid, were able to reach the cotyledonary stage. See Paques et al., Acta. Hort. 319:95-100, 1992. They found that maturation of the conifer embryos could not be achieved when the embryos were placed directly in a liquid medium. Specifically, embryos in contact with the liquid medium failed to develop. Vagner et al. in Hvoslef-Eide A. K. and W. Preil (eds.) Liquid culture systems for in vitro plant propagation. Springer. Netherlands, pages 389-402, 2005, reported that cultivation of Norway spruce embryogenic cell lines in liquid embryo development medium resulted in severe decrease in the number of developed embryos compared to development on gelled or an intermediate raft system.
Ingram and Mavituna, Plant Cell Tiss. Org. Cult. 61:87-96. 2000, found that bioreactor type can influence proliferative growth of Picea embryogenic cultures, although they did not examine embryo development directly in liquid culture bioreactor vessels. Instead, they used a submerged culture system whereby proliferating cells from the bioreactor were transferred to a layer of liquid embryo development medium overlaying gelled embryo development medium. They found that cotyledonary embryo production was very much reduced, by an average of 88%, in this combined liquid and gelled culture system compared to the gelled culture in one cell line and slightly reduced (by 27%) in another cell line. This liquid-gel combination system does not provide a viable system for large-scale embryo production.
Accordingly, it is established that embryo development in Picea species is generally better if proliferating cell cultures are transferred to gelled embryo development medium, regardless of whether they undergo the proliferation phase in a standard flask system or in a bioreactor system. Neither Pacques (1992) nor Ingram and Mavituna (2000) employ a completely liquid embryo development system.
While it is well established that polyethylene glycol be included in embryo development media, it also is well established that polyethylene glycol can harm embryos. Hence, Hogberg et al., Scand. J. For. Res. 16:295-304; 2001, describe the detrimental effects on germination of conifer somatic embryos matured on gelled, i.e., non-liquid-based, embryo development medium containing PEG. PEG also may be omitted from gelled embryo development medium during the latter phase of embryo development. See also U.S. Patent Nos. 5,731,204 and 5,731,191, which are incorporated herein by reference.
The gelled method, while reducing the detrimental effects of PEG, however, does not lend itself to large-scale production. This is because (1) both the embryo development and the post embryo development phases require a gelled medium, and (2) removal of PEG during embryo development requires manually transferring the tissue to a new gelled medium. Aside from these practical downsides, scaling up of such methods can prove costly and burdensome.
There has been, however, complete development of Norway spruce somatic embryos in liquid medium. See Gorbatenko et al. Int. J. Plant Sci. 162 (6):1211-1218, 2001. Gorbatenko, however, used continuous and prolonged exposure to PEG at high concentrations, e.g., 7.5%, which may have deleterious affects on embryo regeneration efficiency.
A bioreactor system for maturation of conifer somatic embryos that utilizes a tissue immobilization phase following a submerged tissue phase is described in U.S. Patent Application 2005/0287660, which is incorporated herein by reference. This two phase system is complex and requires more manipulation and regulation of the tissue compared to a completely liquid embryo development system as described by our invention. Thus, this bioreactor system does not achieve the advantages afforded by a completely liquid embryo development system as obtained in our invention.
Typically, therefore, conventional embryo development methods (i) use gelled media and are therefore not readily amenable to large-scale production requirements, (ii) use a partial liquid/gel method where embryos are placed onto a surface that is saturated with liquid media, (iii) use liquid-based bioreactors where the liquid media passes under embryos that are in contact with, but elevated above, the passage of liquid, (iv) use polyethylene glycol at concentrations that are known to have detrimental effects on embryo development. Hence, conventional methods for developing embryos can be encumbered by one or more of these parameters or limitations.
The present inventive “all-liquid” method avoids these drawbacks. In this method, embryos develop entirely within the desired volume of liquid without direct exposure to the atmosphere. Further, where polyethylene glycol is incorporated into the liquid medium it is (a) at leyels below which are normally used, (b) only temporarily present in the liquid medium, or (c) gradually increased or decreased in concentration at the will of the operator. An added benefit of the presently inventive method is that it increases the numbers of germination-competent embryos that can be produced per volume of liquid media and thereby accelerates the plant production potential and concomitantly decreases handling costs and manual labor costs, as well as the embryo production costs.